Meiotic sex chromosome inactivation (MSCI) during spermatogenesis is characterized by transcriptional silencing of genes on both the X and Y chromosomes in mid to late pachytene spermatocytes. MSCI is believed to result from meiotic silencing of unpaired DNA because the X and Y chromosomes remain largely unpaired throughout first meiotic prophase. Thus, unlike X-chromosome inactivation in female embryonic or somatic cells, where 25-30% of X-linked structural genes have been reported to escape inactivation, there have been no previous reports of genes that escape the silencing effects of MSCI in primary spermatocytes. However, we recently discovered that many X-linked microRNAs (miRNAs) are transcribed and processed in pachytene spermatocytes. This unprecedented escape from MSCI suggests that these miRNAs participate in one or more critical functions at this stage of spermatogenesis. This is corroborated by our preliminary finding that chimeric mice carrying a high percentage of cells bearing a knockout of a major cluster of X-linked miRNA genes display a fertility defect manifest as a block during the meiotic phase of spermatogenesis. This is significant because despite recent reports describing expression of an abundance of miRNAs and other small, non-coding RNAs during spermatogenesis, essentially nothing is known about the function of any of these. In this application, we first propose to investigate the molecular mechanism by which these X-linked miRNA genes escape MSCI (Aim 1). We then propose to test two hypotheses (which are not mutually exclusive) regarding function of these miRNAs, including their role as post- transcriptional regulators of autosomal mRNAs that are synthesized during meiosis but not translated until the postmeiotic period (Aim 2), and their role in regulating the process of MSCI itself (Aim 3). This study is highly novel in that it is designed to reveal an unprecedented mechanism of escape from MSCI, and to identify actual functions of those X-linked miRNAs that undergo this escape during spermatogenesis. PUBLIC HEALTH RELEVANCE: Understanding the molecular and genetic mechanisms by which sperm are produced is critically important for diagnosis and treatment of male infertility, as well as for the development of non-hormonal male-specific contraceptives. Data from the present study will contribute insight into the function of X-linked miRNAs in the control of sperm production and male fertility.